엽록체 항산화기구 대사조절에 의한 환경스트레스 내성 식물

권석윤,이영표,임순,이행순,곽상수,Kwon Suk-Yoon,Lee Young-Pyo,Lim Soon,Lee Haeng-Soon,Kwak Sang-Soo 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.3

Injury caused by reactive oxygen species (ROS), known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Chloroplasts are specially sensitive to damage by ROS because electrons that escape from the photosynthetic electron transfer system are able to react with relatively high concentration of $O_2$ in chloroplasts. To cope with oxidative stress, plants have evolved an efficient ROS-scavenging enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), and low molecular weight antioxidants including ascorbate, glutathione and phenolic compounds. To maintain the productivity of plants under the stress condition, it is possible to fortify the antioxidative mechanisms in the chloroplasts by manipulating the antioxidation genes. A powerful gene expression system with an appropriate promoter is key requisite for excellent stress-tolerant plants. We developed a strong oxidative stress-inducible peroxidase (SWPA2) promoter from cultured cells of sweetpotato (Ipomoea batatas) as an industrial platform technology to develop transgenic plants with enhanced tolerance to environmental stress. Recently, in order to develop transgenic sweetpotato (tv. Yulmi) and potato (Solanum tuberosum L. cv. Atlantic and Superior) plants with enhanced tolerance to multiple stress, the genes of both CuZnSOD and APX were expressed in chloroplasts under the control of an SWPA2 promoter (referred to SSA plants). As expected, SSA sweetpotato and potato plants showed enhanced tolerance to methyl viologen-mediated oxidative stress. In addition, SSA plants showed enhanced tolerance to multiple stresses such as temperature stress, drought and sulphur dioxide. Our results strongly suggested that the rational manipulation of antioxidative mechanism in chloroplasts will be applicable to the development of all plant species with enhanced tolerance to multiple environmental stresses to contribute in solving the global food and environmental problems in the 21st century.

엽록체 항산화기구 대사조절에 의한 환경스트레스 내성 식물

권석윤,곽상수,임순,이행순,이영표 한국식물생명공학회 2005 JOURNAL OF PLANT BIOTECHNOLOGY Vol.32 No.3

Frankia sp. strain SNU 014201의 nif-H, D, K, 유전자 클로닝

권석윤,강명수,안정선 한국미생물학회 1992 미생물학회지 Vol.30 No.1

물오리나무의 뿌리혹에서 분기한 Frankia sp. SNU 014201 공생균주의 게놈내에 13.5 kb의 EcoRI, 18.0 kb 의 BamHI, 10.5 kb의 Bg/II, 4.5 kb의 KpnI 절편에 nif-H, D 유전자가 존재함을 확인하였다. 람다 파아지 EMBI3-BamHI arm을 사용하여 제조한 genomic library 에서 nif유전자를 포함하고 있는 14개의 재조합 파아지 클론을 선별하였다. 이들 중 Ahnif-12번 클론은 nif 유전자를 포함하고 있는 18kb 의 삽입 DNA 를 가지고 있었으며, 이중 7.9 kb 의 BamHI 절편내에 nif-H. D. K가 3.6kb 의 HindIII/KpnI 절편내에 nif D 의 일부와 H 가 위치하고 있었다. 따라서 이등 절편을 각각 subcloning 하고 제한효소 지도를 작성한 결과, Frankia sp. SNU 01420의 nif-H. D. K 유전자는 6.5 kb 의 Hind III/Bam HI 절편과 5.2 kb Sal/IBamHI 절편내에 연속 배열하고 있다. nif (nitrogen fixation)-H.D, K genes of Frankia sp. SNU 014201. a symbiotic strain isolated from root nodule of Alnus hirsura, were found to be located in the genome on 13.5 kb of EcoRI, 18.0 kb of BamHI, 10.5 kb of BglII and 4.5 kb of KpnI fragments. Using EMBL-3 BamHI arms of bacteriophage lambda. the genomic library was constructed. from which fourteen recombinant phage nif-clones were selected. Among them, Ahnif-I2 had insert DNA of 18 kb, in which 7.9 kb of BamHl fragment contained nif-H, D, K and 3.6 kb of HindlIl/KpnI had nif-H and partial -D. Therefore, the 7.9 kb and 3.6 kb fragments were subcloned and partial restriction maps were constructed. As the results, nif-F1, D.K genes were found to be located continuously on the 6.5 kb of HindII/BamHI and 5.2 kb of SalIIBamHI fragment in the genome of Frankia sp. SNU 014201.

물오리나무의 뿌리혹으로부터 Frankia 홍생규주의 (洪生菌株) 분리

권석윤(Seok Yoon Kwon),안정선(Chung Sun An) 한국식물학회 1989 Journal of Plant Biology Vol.32 No.1

An endophyte was isolated from the root nodule of Alnus hirsuta. The isolated endophyte was identified as a Frankia sp. through morphological characteristics. Their infectivity and effectivity were confirmed by introgen-fixing root nodules induced on inoculated Alnus seedlings. Reisolated endophyte from the induced nodule showed identical morphological characteristics as the first isolate, showing the nodule was induced by the first isolate. Consequently, the first isolate was confirmed as a true symbiont of Alnus hirsuta root nodule. The isolate was designated as a Frankia SNU 014201 strain.

양배추 RNA-Seq 데이터를 이용한 full length transcript assembly

김상미,최준경,이봉우,권석윤,김혜란,조성환 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

고품질의 표준 유전자 세트를 확보하는 것은 작물의 기능연구 수행에 매우 중요하다. 고품질의 표준 유전자 세트란 형성된 transcripts의 assembly의 정확성이 높고, full length transcript의 비율이 높은 유전자 세트를 뜻한다. 본 연구에서는 표준 유전체가 보고되지 않은 양배추의 고품질 표준 유전자 세트 확보를 목표로 하며, 그 중 full length transcript 비율이 어느 정도 인지를 측정하고자 한다. 본 연구에서 full length transcript는 표준 유전체에 90% 이상 일치하며, 전사체의 5’UTR과 3’UTR 모두 가진 transcript로 정의하고 분석을 수행하였다. 우선 표준 유전체가 있는 애기장대를 이용하여 full length transcript의 비율 측정하였다. de novo 어셈블리로 애기장대의 표준 유전자 세트를 작성한 결과 26,255 transcripts가 형성되었으며, 26,084 transcripts가 표준 유전체에 90% 이상 일치됨을 확인했다. 그 중 91%에 해당하는 transcripts는 알려진 전사체 13,852개와 매칭이 되었는데, 43%에 해당하는 10,231 transcripts가 전사체 13,852개의 full length transcript로 확인되었다. 위와 동일한 방법을 적용하여 양배추의 표준 유전자 세트를 형성한 결과 53,562 transcripts를 형성되었다. 양배추는 표준 유전체의 부재로 알려진 식물 30종의 아미노산 서열을 이용하여 분석하였다. 그 결과 35,273 transcripts가 알려진 전사체와 매칭이 되었으며, 그 중 45%에 해당하는 15,765 transcripts가 full length transcripts임을 확인하였다. 이 비율은 이미 보고된 유칼립투스와 제브라피시의 transcriptome assembly를 통해 얻어진 full length transcripts 6,208(39.5%), 9,625(26%) 보다 더 향상된 수준이다. 본 연구에서 수행된 방법론을 다른 작물의 표준 유전자 세트 형성에 적용하였을 때 보다 향상된 고품질 표준 유전자 세트를 생산할 것으로 예측된다.

인체 락토페린 생산 형질전환 가시오갈피 배양세포

조승현,권석윤,김재훈,이기택,곽상수,이행순,Jo Seung-Hyun,Kwon Suk-Yoon,Kim Jae-Whune,Lee Ki-Teak,Kwak Sang-Soo,Lee Haeng-Soon 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.3

락토페린은 철 결합 당단백질로서 항 미생물활성과 면역강화와 같은 생리활성 기능을 가지고 있다. 본 연구는 배양 세포 고 발현 SWPA2 promoter를 이용하여 인체락토페린(hLf)을 생산하는 형질전환 가시오갈피 배양세포주 개발에 관한 것이다. 형질전환에 이용된 벡터는 산화스트레스 유도성 SWPA2 promoter의 조절 하에서 hLf이 소포체로 targeting되도록 제작된 SWPA2pro::ER-hLf/pCAMBIA이다. hLf을 생산하는 각 형질전환 배양세포들은 PCR과 Southern분석을 통해 hLf 유전자가 가시오갈피 게놈내로 성공적으로 도입되었음을 확인하였으며, western blot과 ELISA를 통해 형질전환 가시오갈피 배양 세포주에서 hLf 단백질이 활성이 있음을 확인하였다. 형질전환 가시오갈피 배양세포에서 hLf 단백질의 함량은 세포배양이 진행될수록 증가하여 정지기 때 가장 높았으며 전체 수용성 단백질의 약 3%를 차지하였다. 따라서 본 연구에서 개발된 인체락토페린을 고생산하는 약용식물 가시오갈피 배양 세포주는 산업적으로 이용될 수 있을 것으로 기대된다. Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.

CuZnSOD와 APX를 엽록체에 발현시킨 담배식물체의 Highlight와 Chilling 스트레스에 대한 광합성 효율

김윤희,권석윤,방재욱,곽상수,Kim, Yun-Hee,Kwon, Suk-Yoon,Bang, Jae-Wook,Kwak, Sang-Soo 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.4

환경스트레스에 대한 항산화효소의 보호효과를 이해하고 효과적인 스트레스내성 식물을 개발하기 위하여 CuZnSOD와 APX를 엽록체에 발현시킨 형질전환식물체 (CA식물체)에 강한 빛 (highlight, 1,100$\mu$mol m$^{-2}$ sec$^{-1}$)과 4$^{\circ}C$ chilling을 처리하였다. CA식물체 잎절편에서의 보호효과를 CuZnSOD을 도입한 SOD식물체, APX을 도입된 APX식물체 및 비형질전환식물체 (NT식물체)의 것과 비교하였다. CA식물체의 광계2에서의 광합성 효율 (Fv/Fm)은 highlight와 4$^{\circ}C$ chilling복합처리 1시간째에서 NT식물체에 비해 약 15%의 보호효과를 나타내었고, 광계1에서의 P700 redox state는 처리 후 3시간째에 약 23%의 보호효과를 나타내었다. SOD식물체와 APX식물체의 복합처리에 대한 보호효과는 CA식물체와 NT식물체의 중간 효과를 나타내었다. 이러한 결과는 엽록체에서 CuZnSOD와 APX의 동시발현이 highlight와 chilling에 의한 산화스트레스를 극복하는 데 매우 중요하게 관여함을 직접적으로 제시하는 것이다. In order to understand the protection effects of antioxidant enzymes against oxidative stress caused by various environmental stresses, transgenic tobacco (Nicotiana tabacum cv, Xanthi) plants expressing both copper/zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) in chloroplasts (referred to as CA plants) were subjected to highlight (1,100$\mu$mol m$^{-2}$ sec$^{-1}$) and chilling at 4$^{\circ}C$. The protection effects of CA plants using leaf discs were compared with those of transgenic plants expressing either CuZnSOD or APX in chloroplasts (SOD plants or APX plants, respectively) and non-transgenic (NT) plants. CA plants showed about 15% protection in the photosynthetic efficiency (Fv/Fm) of photosystem II relative to NT plants 1 hr after treatment of both highlight and chilling, whereas they showed about 23% protection in the redox state of P700 in photosystem I at 3 hr after treatment. SOD plants or APX plants showed an intermediate protection effect between CA plants and NT plants. These results demonstrated that the coexpression of CuZnSOD and APX in chloroplasts importantly involves in the protection effects against oxidative stress caused by various environmental stresses.

인체 락토페린 생산 형질전환 가시오갈피 배양세포

조승현,권석윤,이기택,김재훈,곽상수,이행순 한국식물생명공학회 2005 JOURNAL OF PLANT BIOTECHNOLOGY Vol.32 No.3

락토페린은 철 결합 당단백질로서 항 미생물활성과 면역 강화와 같은 생리활성 기능을 가지고 있다. 본 연구는 배양 세포 고 발현 SWPA2 promoter를 이용하여 인체락토페린(hLf)을 생산하는 형질전환 가시오갈피 배양세포주 개발에 관한 것이다. 형질전환에 이용된 벡터는 산화스트레스 유도성 SWPA2 promoter의 조절 하에서 hLf이 소포체로 targeting되도록 제작된 SWPA2pro::ER-hLf/pCAMBIA이다. hLf 을 생산하는 각 형질전환 배양세포들은 PCR과 Southern 분석을 통해 hLf 유전자가 가시오갈피 게놈내로 성공적으로 도입되었음을 확인하였으며, western blot과 ELISA를 통해 형질전환 가시오갈피 배양 세포주에서 hLf 단백질이 활성이 있음을 확인하였다. 형질전환 가시오갈피 배양세포에서 hLf 단백질의 함량은 세포배양이 진행될수록 증가하여 정지기 때 가장 높았으며 전체 수용성 단백질의 약 3%를 차지하였다. 따라서 본 연구에서 개발된 인체락토페린을 고생산하는 약용식물 가시오갈피 배양 세포주는 산업적으로 이용될 수 있을 것으로 기대된다. Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protectionagainst microbial and virus infection, stimulation of the imune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein folowing Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cel cultures to produce hLf protein.

An insight into the tomato spotted wilt virus (TSWV), tomato and thrips interaction

GUPTARAVI,권석윤,김선태 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.3

Lycopersicum esculentum (tomato), is a member of the Solanaceae family with a global production of approximately 159 million tons per year. However, the productivity of tomato is constrained by many factors including biotic and abiotic stresses. Of these, tomato spotted wilt virus (TSWV) is one of the major threats to tomato productivity. TSWV is a ribovirus and is transmitted by small insects commonly known as thrips. Several approaches have been utilized in the past few decades to understand the plant and thrips responses against TSWV. These include conventional molecular biology to high-throughput genomics, transcriptomics, and proteomics approaches which have led to the identification of several genes/proteins involved in the tomato/thrips-TSWV interaction. Moreover, several genes (such as Sw-1a and Sw-1b, sw-2, sw-3, sw-4, Sw-5, Sw-6, and Sw-7) and proteins (like DNA-J) have also been identified from its plant hosts which provide resistance against this deadly virus. In this mini-review, we are summarizing the progress made so far in this area to provide the overview of tomato, thrips and TSWV interaction.

Ectopic Expression of a Cold-Responsive CuZn Superoxide Dismutase Gene, SodCc1, in Transgenic Rice (Oryza sativa L.)

이상춘,권석윤,김성룡 한국식물학회 2009 Journal of Plant Biology Vol.52 No.2

A cytosolic antioxidant enzyme gene, SodCc1, encoding CuZn superoxide dismutase was characterized from rice. SodCc1 mRNA was up-regulated by cold (4°C and 12°C) and by abscisic acid (ABA) treatment. Transgenic rice plants of Ubi:SodCc1 were generated and overexpression of SodCc1 was confirmed at both transcriptional and translational levels. A stress tolerance test via chlorophyll fluorescence at the seedling stage showed no enhanced tolerance by Ubi:SodCc1 plants to cold or methyl-viologen-induced oxidative stress, but they were slightly resistant to drought. Our wilting assay demonstrated no improvement in tolerance to either cold or drought, indicating that cytosolic SodCc1 might not be significantly involved in conferring such tolerances in rice.